Hybridization Approach to Identify Salicylanilides as Inhibitors of Tubulin Polymerization and Signal Transducers and Activators of Transcription 3 (STAT3)

The superimposition of the X-ray complexes of cyclohexanediones (i.e., TUB015), described by our research group, and nocodazole, within the colchicine binding site of tubulin provided an almost perfect overlap of both ligands. This structural information led us to propose hybrids of TUB015 and nocodazole using a salicylanilide core structure. Interestingly, salicylanilides, such as niclosamide, are well-established signal transducers and activators of transcription (STAT3) inhibitors with anticancer properties. Thus, different compounds with this new scaffold have been synthesized with the aim to identify compounds inhibiting tubulin polymerization and/or STAT3 signaling. As a result, we have identified new salicylanilides (6 and 16) that showed significant antiproliferative activity against a panel of cancer cells. Both compounds were able to reduce the levels of p-STAT3Tyr705 without affecting the total expression of STAT3. While compound 6 inhibited tubulin polymerization and arrested the cell cycle of DU145 cells at G2/M, similar to TUB015, compound 16 showed a more potent effect on inhibiting STAT3 phosphorylation and arrested the cell cycle at G1/G0, similar to niclosamide. In both cases, no toxicity towards PBMC cells was detected. Thus, the salicylanilides described here represent a new class of antiproliferative agents affecting tubulin polymerization and/or STAT3 phosphorylation.


Introduction
Compounds binding in the colchicine site at the αβ-tubulin interface have been extensively studied as antimitotic agents, although their therapeutic applications could be much wider than their effect on mitosis [1]. Colchicine (1, Figure 1) is used for the treatment of gout and familial Mediterranean fever [2]. Its anti-inflammatory properties have led to its inclusion as a therapeutic agent for COVID-19 [3], although its use outside clinical trials has been discouraged [4]. The natural stilbene combretastatin A-4 (CA-4, 2), which also binds to tubulin at the colchicine site, has been shown to have vascular disrupting activity in the tumor microenvironment and its phosphate prodrug fosbretabulin has progressed to phase III clinical trials [5]. The therapeutic interest related to compounds binding at the colchicine site in tubulin has made these compounds an extremely active area of research, and different chemical classes have been described [6][7][8]. Chemical structures of colchicine-site binders in tubulin (1)(2)(3)(4)(5) and different salicylanilides (6)(7)(8)(9) including niclosamide (7) and the here addressed compounds (6,9).
Our group has been involved in the identification and optimization of new scaffolds that are able to bind at the colchicine site in tubulin. Performing a ligand-based virtual screening approach, using TN-16 (3) as the model compound, we identified a series of cyclohexanediones that are potent colchicine-site binders [9]. Recently, the binding mode of these cyclohexanediones [i.e., TUB015 (4), Figure 1] into the colchicine site in tubulin has been determined by X-ray diffraction [10]. As expected, the compound binds in a similar way to TN-16 into the deeper part of the β-subunit (Figure 2A). The 5-phenyl ring of the cyclohexanedione is lodged into a cavity lined by residues βIle4, βTyr52, βGln136, βPhe169, βTyr202, βThr239, βLeu242, and βLeu252, among others, while there is a polar interaction between one of the carbonyls of the cyclohexanedione and the side chain of βGlu200. On the other hand, the 2-methoxyaniline of TUB015 is stabilized by hydrophobic interactions. Among the published structures of colchicine ligands with tubulin, nocodazole (5, Figure 1) occupies the same deep pocket as TUB015, and, interestingly, it also interacts with βGlu200 ( Figure 2B) [11]. Indeed, when the binding modes of TUB015 and nocodazole are superimposed, there is almost a perfect fit, as shown in Figure 2C. (A) X-ray complex structure of TUB015 (4, magenta sticks) in tubulin (α-tubulin shown in blue marine and β-tubulin in cyan). (B) X-ray complex structure of nocodazole (5, violet sticks) in tubulin (α-tubulin is shown in orange and β-tubulin in light orange). (C) Superimposition of X-ray structures of TUB015 (4) and nocodazole (5). Selected residues of the binding pocket are shown in sticks and labeled. Dashed lines represent hydrogen bonds.
In our aim to identify new scaffolds able to bind at the colchicine binding site of tubulin, we designed the hybrid molecule 6 (Figure 1), carrying a methyl carbamate as in nocodazole, an OH able to interact with βGlu200, and a 2-methoxyaniline as in TUB015. The resulting molecule 6 is a salicylanilide that can be structurally considered as a niclosamide analog. Niclosamide (7) is extensively studied for anticancer purposes affecting different pathways [12,13]. Among the described activities, niclosamide has been reported to inhibit the cytoplasmic signal transducer and activator of transcription-3 (STAT3) signaling [14,15], a pathway regulating important processes for tumorigenesis such as cell proliferation, cell cycle progression, or apoptosis. While in normal cells the levels of STAT3 remain transient, in a high number of solid tumors and hematological cancers, STAT3 is constitutively active [16]. Cytoplasmic STAT3 is activated through the phosphorylation at residue Tyr705 by Janus associated kinase (JAK) or other kinases, leading to its dimerization and translocation into the nucleus where STAT3 activates the transcription of a wide variety of genes related to cell proliferation and differentiation, immune response, or apoptosis among others [17]. Very recently, niclosamide has been shown to increase the efficacy of PD-1/PD-L1 immune checkpoint by the blockage of phosphorylated STAT3 to the promoter of PD-1 [18], opening new possibilities for cancer immunotherapy through targeting STAT3 [19].
Other salicylanilides based on hydroxynaphthalene carboxamides have also been recently described as STAT3 inhibitors [20]. Interestingly, in this naphthalene series, the best results were obtained with compound 8 (Figure 1), carrying a 3,5-diCF 3 aniline. Thus, we hypothesized that compounds with the general structure 9, with one or two substituents (R 1 and R 2 ) at the aniline moiety, including a 3,5-diCF 3 substitution, may constitute a new class of antiproliferative agents by combining tubulin-binding at the colchicine site (similar to TUB015) and STAT3 inhibition based on the reported activities for niclosamide and hydroxynaphthalene derivatives exemplified by compound 8. Additionally, the importance of the phenolic OH in this series has been studied by its replacement with F (9, X = F). Besides the synthesis of this new family of salicylanilides, their antiproliferative activity against a panel of cancer cell lines is reported here. Moreover, additional assays have been performed to propose tubulin and STAT3 signaling as relevant targets that account for the antiproliferative effects.

Synthesis
The synthesis of the hybrid molecule 6 was addressed by reaction of 4-amino-2hydroxybenzoic acid (10) with methyl chloroformate in ethyl acetate as reported in other hydroxyanilines [21] to provide a 95% yield of carbamate 11 (Scheme 1). Further reaction of 11 with o-anisidine was performed by heating in toluene to 150 • C under MW irradiation in the presence of PCl 3 [22] to yield the target compound 6. Similarly, acid 11 reacted with m-or p-anisidine (PCl 3 , toluene, 150 • C, MW) to afford the salicylanilides 12 and 13 in 43 and 35% yields, respectively, where the methoxy group is located at positions 3 or 4 in the aniline moiety. In the cyclohexanedione series exemplified by TUB015, the 2-OMe could be replaced by a 2-Cl without a significant loss in antiproliferative activity [9]. Thus, a chlorine substituent was also introduced at positions 2 or 3 of the aniline in the salicylanilide series (compounds 14 and 15). Compound 14 was obtained by reaction of 11 with 2-chloroaniline in the presence of N,N -dicyclohexylcarbodiimide in DMF [23] after 20 min of irradiation in the MW at 100 • C, while compound 15 was obtained by the standard procedure with PCl 3 . Based on the antiproliferative activity and mechanism of action described for compound 8 [20], the synthesis of 16 was addressed by reaction of 11 with the 3,5-bis-(trifluoromethyl)aniline using PCl 3 . Furthermore, the 3,5-bis-fluoro derivative 17 was synthesized following the same approach. As will be later discussed, compound 16 afforded potent antiproliferative activity; thus, the monosubstituted compound with a CF 3 at position 3 (compound 18) was also synthesized. In all cases (6,(12)(13)(14)(15)(16)(17)(18), the 1 H NMR spectra registered in DMSO-d 6 showed the presence of a broad singlet at 11.66-12.31 ppm corresponding to the phenol, as described for other salicylanilides. [24] Finally, in order to study the influence of the hydroxyl substituent of 6, the synthesis of the corresponding fluorinated derivative was accomplished. The reaction between 2-fluoro-4-aminobenzoic acid (19) and methyl chloroformate provided the acid 20, which further reacted with oanisidine to give the benzamide 21 in 66% yield. It should be mentioned that the 1 H NMR spectrum of compound 21 showed the signal corresponding to the NH of the amide as a doublet, with a coupling constant of 11 Hz. By recording a fluorine-decoupled 1 H NMR spectrum, this signal was observed as a broad singlet, indicating that the splitting is due to a long-range coupling with the fluorine (see Supplementary Materials). This splitting has been reported in similar compounds [25,26].

Antiproliferative Activity
The synthesized compounds were evaluated against a panel of cancer cell lines, as shown in Table 1. Compounds TUB015, niclosamide, and nocodazole were included as reference compounds. Compound 6 showed marked antiproliferative activity against the different cancer cell lines tested, either carcinoma or leukemia cells, with IC 50 values in the low µM range. Moving the methoxy substituent to position three of the aniline (12) resulted in an inactive compound, while the incorporation of the same substituent at position four (13) led to moderate antiproliferative activity. A similar trend was observed with the chloro substitution in the aniline, with the 2-Cl derivative (14) showing antiproliferative activity in the low µM range while the 3-Cl derivative (15) was inactive. Interestingly, the salicylanilide bis-substituted with a trifluoromethyl group at positions 3 and 5 (16) also showed clear antiproliferative activity against all the cell lines tested with IC 50 values in the low µM range, while the 3,5-diF compound (17) was barely active against most cell lines tested, except for the HL-60 cell line. The monosubstitution with a CF 3 at position 3 (18) led to a significantly less active compound than the 3,5-disubstituted analog 16. Finally, the replacement of the phenolic OH in compound 6 by an F atom (21) resulted in the annulation of the antiproliferative activity.
Thus, compounds 6 and 16 were selected for additional assays in order to gain insights into their mechanism of action as antiproliferative agents.

Inhibition of Tubulin Polymerization
In order to determine whether the observed antiproliferative activity was due to interaction with tubulin, compounds 6 and 16, as well as controls TUB015, niclosamide, and nocodazole were tested in a cell-free tubulin polymerization assay, as shown in Figure 3A. Polymerization of tubulin is followed by fluorescence enhancement caused by the incorporation of a fluorescent reporter into the microtubules as polymerization occurs. Without treatment (DMSO control, dashed line), tubulin subunits assemble to form microtubules in a time-dependent manner. Microtubule-destabilizing agents TUB015 and nocodazole prevent tubulin polymerization in a clear dose-dependent manner, with the highest concentration tested (10 µM) completely blocking polymerization ( Figure 3A). Compound 6 exhibited a similar dose-dependent effect on tubulin polymerization confirming that compound 6 directly inhibited tubulin polymerization. The highest dose tested (100 µM) fully suppressed polymerization, and 30 and 10 µM also inhibited the polymerization almost completely. At 3 µM, compound 6 slowed down the rate of the polymerization but could not completely suppress it. The IC 50 value of the effect of compound 6 in tubulin polymerization (3.9 µM) was comparable to those obtained for the positive control compounds TUB015 and nocodazole ( Figure 3B). Compound 16 did not show a significant effect on tubulin polymerization, providing curves similar to the DMSO control or the negative control drug niclosamide. Moreover, immunofluorescence staining of α-tubulin in HEp-2 cells illustrated that salicylanilide 6 disrupts tubulin polymerization in a dose-dependent manner, similarly to TUB015 or nocodazole (see Supplementary Figure S1), further supporting that compound 6 promotes tubulin disassembly. Docking studies performed with compound 6 using the coordinates of the TUB015-tubulin complex indicated that the compound fits in this binding site (see Supplementary Figure S2).

Antiproliferative Activity in Human Prostate Cancer Cells DU145
As mentioned in the introduction, niclosamide and other recently reported salicylanilides (exemplified by compound 8) have been shown to inhibit STAT3 signaling. In order to determine if our salicylanilides 6 and 16 were also interfering with this signaling pathway, proliferation assays were performed in human DU145 prostate cancer cells where STAT3 is constitutively activated [27]. As shown in Table 2, both compounds were able to considerably inhibit DU145 proliferation as determined by WST-1 analysis; this effect was most pronounced after 48 to 72 h. Compound 16 was significantly more active than compound 6 in this cell line, although both compounds showed IC 50 values higher than those of the reference drug niclosamide.

Effects of Compounds 6 and 16 in Regulating STAT3 Signaling Pathway In Vitro
To investigate the effect of compounds 6 and 16 on the regulation of STAT3 signaling, the phosphorylation of STAT3 at Tyr705 was measured in DU145 cells using niclosamide as a positive control. The levels of STAT3 and p-STAT3 Tyr705 were examined by immunoblot after 24 h or 48 h of treatment to better describe the kinetics of phosphorylation changes induced by the compounds under study. Both compounds were tested at concentrations ranging from 1 to 20 µM for a 24 h incubation period and from 0.5 to 10 µM for a 48 h incubation period, while niclosamide was used at a fixed concentration of 5 µM. Compounds 6 and 16 markedly reduced phosphorylation of STAT3 at Tyr705 in a concentration-dependent manner both after 24 h ( Figure 4A) or after 48 h ( Figure 4B), while the total expression of STAT3 was not affected. The levels of p-STAT3 Tyr705 were quantified and represented as fold-change relative to control in Figure 5. After treatment with either compound 6 or 16, p-STAT3 Tyr705 phosphorylation decreased with time of exposure and concentration of the tested compounds. At both time points (24 h, Figure 5A or 48 h, Figure 5B), the effect on STAT3 phosphorylation was more significant for compound 16 than for 6. Indeed, the reduction of the levels of p-STAT3 Tyr705 after 48 h of treatment with compound 16 at 10 µM was very relevant, being quite comparable to the effect observed with niclosamide at 5 µM. Thus, we may conclude that both compounds are able to reduce STAT3 phosphorylation, and in this way, they might affect STAT3 signaling. The levels of the phosphorylated form (p-STAT3 Tyr705 ) were detected by immunoblot analysis using an appropriate antibody. Samples treated with 5 µM of niclosamide (Nic) for 24 h and 48 h were used as positive controls. Summary data of p-STAT3 Tyr705 levels, where the values shown are means ± SD of three independent experiments. The levels of p-STAT3 Tyr705 protein are expressed in relative units following normalization for the intensity of the corresponding β-actin bands. * p < 0.05, ** p < 0.01, *** p < 0.001, significantly different from drug-free control.

STAT3 Translocation Experiments
Once phosphorylated, STAT3 forms dimers that are translocated to the cell nucleus, where it binds to specific DNA sequences and induces transcription of the target genes [28]. It has been described that niclosamide inhibits the step of phosphorylation of STAT3 and reduces STAT3 nuclear translocation [14]. Our results showed that compound 16, similarly to niclosamide, suppressed the EGF-induced nuclear translocation of STAT3 in DU145 cells (Figure 6), while the total amount of cellular STAT3 was not affected by any of the tested compounds. Interestingly, only slight suppression of STAT3 nuclear translocation was observed upon treatment by 20 µM of compound 6. Thus, we can assume that salicylanilide 16, similar to niclosamide, might interfere with STAT3 signaling by suppressing its translocation into the cell nucleus. Since the tested compounds showed an antiproliferative effect in DU145 cells, we further investigated how they affected progression through cell cycle phases. As seen in Figure 7A, compound 6 at 2.5 µM induced accumulation of cells in the G2/M phase as described for TUB015 and similarly to other colchicine site binders [9]. However, compound 16 increased the proportion of cells in G1/G0 phases together with a decrease in the percentage of cells in the S phase ( Figure 7B). Similarly, exposure of cells to niclosamide also led to cell accumulation in the G1/G0 phase of the cell cycle ( Figure 7C).

Analysis of Apoptosis Induction in PBMC
Compounds 6, 16, TUB015, nocodazole, and niclosamide were tested in peripheral blood mononuclear cells (PBMC) in order to evaluate their selectivity toward non-cancerous cells. Compounds 6 and 16 were tested at five different concentrations ranging up to 100 µM, while for the rest of the compounds, the highest concentration tested was 10 µM. As shown in Figure 8 compound 6 did not affect the viability of normal cells, in line with the profile seen for the other microtubule targeting agents TUB015 and nocodazole. For compound 16, an increase in the number of dead PBMCs is detected only at the highest concentration tested (100 µM). Still, this slightly toxic effect is lower than that observed with niclosamide at 10 or even 3 µM. These results indicate that compounds that mainly target microtubules showed almost no impact on the percentages of live cells even at high concentrations, while those acting as STAT3 inhibitors showed a higher proportion of dead and apoptotic cells.  Analytical TLC was performed on silica gel 60 F 254 (Merck, Dramstand, Germany)precoated plates (0.2 mm). Spots were detected under UV light (254 nm) and/or charring with ninhydrin or phosphomolybdic acid.

Chemistry Procedures
Separations on silica gel were performed by preparative centrifugal circular thin-layer chromatography (CCTLC) on a Chromatotron R (Kiesegel 60 PF 254 gipshaltig Merck), with a layer thickness of 1 and 2 mm and a flow rate of 4 or 8 mL/min, respectively.
The following is the general procedure for the reaction of 2-hydroxybenzoic acids with anilines (General procedure A): the appropriate aniline (1.0-2.0 mmol) in toluene (5-10 mL), PCl 3 (0.5-1.0 mmol) was added to a microwaveable vial containing the corresponding 2-hydroxybenzoic acid (1.0 mmol). The reaction vessel was sealed and heated in a microwave reactor at 150 • C for 10 min. The reaction mixture was diluted with DCM (20 mL) and washed with a saturated solution of NH 4 Cl (10 mL). The organic layer was dried over Na 2 SO 4 , filtered, and evaporated to dryness. The residue was purified by CCTLC in the Chromatotron as specified.

Cell Culture and Reference Compounds
Cancer cell lines Capan-1, HCT-116, NCI-H460, HL-60, K-562, and Z-138 were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The DND-41 cell line was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ Leibniz-Institut, Brunswick, Germany). All cell lines were cultured as recommended by the suppliers. Culture media were purchased from Gibco Life Technologies and supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences, Chicago, IL, USA).
A human prostate cancer cell line DU145 was obtained from ATCC and cultivated in RPMI 1640 supplemented with the antibiotic solution (100 U/mL of penicillin, 100 µg/mL of streptomycin (HyClone, GE Healthcare Life Sciences, Chicago, IL, USA), and 10% fetal bovine serum. Cells were maintained in a humidified incubator with 5% CO 2 at 37 • C and were regularly tested for the presence of mycoplasma contamination.
Tested compounds were dissolved in dimethyl sulfoxide (DMSO) from Sigma Aldrich (St. Louis, MO, USA). The final concentration of DMSO in the assays never exceeded 0.1% (v/v).

Cell Proliferation Assays
Adherent cell lines HCT-116, NCI-H460, and Capan-1 cells were seeded at a density between 500 and 1500 cells per well in 384-well tissue culture plates (Greiner). After overnight incubation, cells were treated with different concentrations of the test compounds. Suspension cell lines HL-60, K-562, Z-138, and DND-41 were seeded at densities ranging from 2500 to 5500 cells per well in 384-well culture plates containing the test compounds at the same concentration points. The plates were incubated and monitored at 37 • C for 72 h in an IncuCyte (Essen BioScience Inc., Sartorius; Göttingen, Germany for real-time imaging of cell proliferation. Brightfield images were taken every 3 h, with one field imaged per well under 10× magnification. Cell growth was then quantified based on the percent cellular confluence, as analyzed by the IncuCyte image analysis software and used to calculate IC 50 values. Compounds were tested in at least two independent experiments and represented as mean ± SEM.

WST-1 Analysis of Cell Proliferation
DU145 cells were seeded at 7 × 10 3 cells/100 µL per well in 96-well plates and allowed to attach to the wells for 24 h. Cells were then treated with various concentrations of compound 6 or 16 to reach the final concentration range of 0.1-30 µM, and with various concentrations of niclosamide to reach the final concentration range of 0.01-10 µM. Cells were then incubated for 24 h, 48 h, or 72 h. Cell proliferation of DU145 cells was determined using Cell Proliferation Reagent WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) (Roche Diagnostics, Mannheim, Germany), as it was previously described [29,30]. WST-1 analysis was performed in three independent experiments, with each condition tested in triplicate. The IC 50 values were determined using the nonlinear regression four-parameter logistic model using GraphPad Prism 5.03 software (GraphPad Software, San Diego, CA, USA).

In Vitro Tubulin Polymerization Assay
In vitro tubulin polymerization was carried out using the fluorescence-based tubulin polymerization assay (BK011P, Cytoskeleton, Denver, CO, USA) as described by the manufacturer. Briefly, half area 96-well plates were warmed to 37 • C 10 min prior to assay start. Test compounds and reference drugs were prepared at 10× stock solutions and added in 5 µL in duplicate wells. Ice-cold tubulin polymerization buffer (2 mg mL −1 tubulin in 80 nM Pipes, 2 mM MgCl 2 , 0.5 mM EGTA, pH 6.9, and 10 µM fluorescent reporter + 15% glycerol + 1 mM GTP) was added into each well, followed by reading with a Tecan Spark fluorimeter in kinetic mode, 61 cycles of 1 reading per minute at 37 • C, 4 reads per well (Ex. 350 nm and Em. 435 nm).

Tubulin Immunofluorescence Staining
HEp-2 cells (human cervix carcinoma) were seeded at 15,000 c/well in 8-well chamber slides (Ibidi). After overnight incubation, cells were treated with test compounds or reference drugs for 3 h and then fixed with 4% PFA, washed, and permeabilized. Standard immunofluorescence procedures were performed using primary mouse anti-alpha tubulin antibody (sc-5286, Santa Cruz Biotechnology) and secondary goat anti-mouse IgG conjugated to Alexa Fluor ® 488 (A11001, Invitrogen). Nuclei were counterstained with DAPI. Images were taken with a Leica TCS SP5 confocal microscope using an HCX PL APO 63x (NA 1.2)/water immersion objective.

Western Blotting
Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethanesulfonylfluoride (PMSF, 1 mM), both from Cell Signaling Technology (Danvers, MA, USA) and protease and phosphatase inhibitor cocktails (Roche Diagnostics, Mannheim, Germany). SDS-PAGE and western blotting were performed according to the previously described protocols [20]. Membranes were stained with anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) to assess equal sample loading. Antibodies against STAT3 (79D7, 4904) and p-STAT3 [Tyr 705] (D3A7, 9145) were purchased from Cell Signaling Technology. The intensity of bands was semiquantitatively analyzed using the ImageJ software (National Institute of Mental Health, Bethesda, MD, USA).
3.2.7. Analysis of STAT3 Nuclear Translocation DU145 cells were serum-starved overnight and treated with the indicated concentrations of compounds 6, 16, and niclosamide for 2 h. After that, the cells were stimulated with 100 ng/mL EGF (Abcam, Cambridge, UK) for 15 min. Nuclear extracts were prepared using Nuclear Extraction Kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. Whole cell lysates were prepared by use of RIPA buffer supplemented with PMSF (1 mM) and protease and phosphatase inhibitor cocktails. DU145 cells were detached with trypsin/EDTA and counted with a hemocytometer. For nuclear extract preparation, cells were centrifuged for 5 min at 1000 rpm, and the supernatant was discarded. The cell pellet was resuspended in 100 µL of 1X Pre-Extraction Buffer per 10 6 cells and incubated on ice for 10 min. Then, samples were centrifuged for 1 min at 12,000 rpm, and the cytoplasmic extract was removed from the nuclear pellet. Extraction Buffer containing DTT and PIC was added to the nuclear pellet (10 µL per 10 6 cells). After 15 min of incubation on ice, samples were centrifuged for 10 min at 14,000 rpm at 4 • C. Then, supernatants were collected, and the protein concentration of the nuclear extracts was measured using Roti ® -Quant universal (Carl Roth, Karlsruhe, Germany) according to the manufacturer's instructions. Western blotting was performed according to the previously described protocol [20].

Cell Cycle Analysis
Sub-confluent DU145 cells were treated and subsequently incubated with indicated concentrations of compounds 6, 16, and niclosamide for 48 h. Cell cycle analysis was performed as it was previously described [29]. The cell cycle distribution was analyzed using a flow cytometer BriCyte-E6 (Mindray, Shenzhen, China). The quantification of cell cycle distribution was carried out using the software ModFit LT™ (Verity Software House, Topsham, ME, USA). A total number of 2 × 10 4 cells were analyzed per sample.

Statistical Analysis
Experimental data are expressed as the mean ± standard deviation (SD). Statistical significance between values was evaluated by one-way analysis of variance (ANOVA) paired with Dunnett's post hoc test using GraphPad Prism 5.00 software (GraphPad Software, San Diego, CA, USA) at levels of * p < 0.05, ** p < 0.01 and *** p < 0.001.

Apoptosis Induction Assay
Buffy coat preparations from healthy donors were obtained from the Blood Transfusion Center in Leuven, Belgium. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Lymphoprep (d = 1.077 g/mL) (Nycomed, Oslo, Norway) and cultured in cell culture medium (DMEM/F12, Gibco Life Technologies, Europe, Merelbeke, Belgium) containing 8% FBS. PBMC were seeded at 28,000 cells per well in 384-well, black-walled, clear-bottomed tissue culture plates containing the test and reference compounds at five different concentrations. Propidium iodide was added at a final concentration of 1 µg/mL, and IncuCyte ® Annexin V Green Reagent was added as recommended by the supplier. The plates were incubated and monitored at 37 • C for 72 h in the IncuCyte ® . Images were taken every 3 h in the brightfield and the green and red fluorescence channels, with one field imaged per well under 10× magnification. Quantification of the fluorescent signal after 72 h in both channels using the IncuCyte ® image analysis software allowed for the calculation of the percentage of live, dead, and apoptotic cells. All compounds were tested in two independent experiments, implying PBMC originated from two different donors.

Conclusions
Molecular hybridization is considered an interesting approach in drug design to identify new compounds that may present dual modes of action while reducing nondesired side effects. Here we have undertaken a double strategy based on the binding mode of our cyclohexanediones, exemplified by TUB015, at the colchicine site in tubulin and its almost perfect overlap with the binding mode of nocodazole, in the search for novel chemical series that inhibit tubulin polymerization and affect STAT3 signaling. First, we have identified a central core of salicylanilide that could replace the cyclohexanedione of TUB015, employing a scaffold hopping strategy, while the functionalization of both ends of this scaffold has been performed following a hybridization approach incorporating a methoxycarbonylamino, present in nocodazole and a 2-methoxyphenylamino, similar to TUB015, resulting in compound 6. Since salicylanilides, including niclosamide, have been reported to inhibit STAT3, different anilines were also assayed to replace the 2methoxyaniline, being particularly relevant for compound 16, functionalized with a 3,5-diCF 3 -anilide. Compounds 6 and 16, which showed antiproliferative activity against a panel of cancer cell lines with IC 50 values in the low µM range, were tested as tubulin polymerization inhibitors (similarly to TUB015) and as STAT3 inhibitors, using niclosamide as a reference compound. Interestingly, compound 6, with a 2-methoxyaniline as the amidine, inhibited tubulin polymerization with IC 50 values close to those of nocodazole or TUB015 and arrested the cell cycle of DU145 cells at G2/M but also showed a moderate effect on inhibiting STAT3 phosphorylation. On the other hand, compound 16, with a 3,5-diCF 3 -anilide, significantly affected STAT3 phosphorylation, inhibited STAT3 nuclear translocation, and arrested the cell cycle at G0/G1 phases, similar to niclosamide, while no effect on tubulin polymerization was observed. Thus, the substitution pattern on the aniline on the same scaffold determines the mechanism of action underlying the antiproliferative activity of this new family of salicylanilides. Interestingly, both compounds did not show significant toxicity to PBMC cells up to 100 µM, while for niclosamide there are dead and apoptotic cells already at concentrations of 1 to 3 µM. The lack of toxicity in PBMC and the involvement of two different and complementary targets in their antiproliferative activity makes these salicylanilides a promising family of anticancer compounds that will be further explored. Moreover, our results also support the general strategy that molecular hybridization may lead to new molecules being able to achieve high efficacy through a multi-target mechanism of action whilst also reducing toxicity.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15070835/s1, Figures S1 and S2: immunofluorescence staining assay of the salicylanilide 6 against α-tubulin in HEp-2 cells and docking of compound 6 in the complex tubulin-TUB015. 1 H and 13 C NMR spectra of the tested compounds.